[The activation of microglia caused by lead and manganese co-exposure induces activation of astrocytes and decrease...

To establish microglia and astrocyte co-culture cell model and investigate the effects of lead and manganese alone or co-exposure on different glia cells and the effect of microglia activation on astrocytes' function.

MTT assay was performed to select the appropriate dose and time of lead and manganese exposure that didn't affect C6 cell growth. Then with BV2 microglia and C6 astrocytes, cell models were established through lead acetate and manganese chloride alone and co-exposure. The cell models were further divided into three types: direct stimulation, conditioned medium and co-culture. In the direct stimulation method, C6 cells were directly treated with complete medium or complete medium containing lead acetate and/or manganese chloride for 24 hours. In conditioned medium method, BV2 cells were cultured in the complete medium or complete medium containing lead acetate and/or manganese chloride for 24 hours, and then the supernatants were centrifuged and used to treat C6 cells for another 24 hours. In co-culture method, BV2 cells were seeded in semipermeable membrane inserts and the inserts were put in a normal 12-well plate; C6 cells were seeded in another 12-well plate; complete medium or complete medium containing lead acetate and/or manganese chloride was added in the wells to culture BV2 cells for 24 hours; the culture medium was abandoned, the cells seeded in the inserts were gently rinsed with complete medium and then the inserts were put into the 12-well plate where C6 were seeded before; the BV2 cells and C6 cells were co-cultured for another 24 hours. The effects of lead and manganese alone or co-exposure on different glia cells were analyzed through CR3/CD11b/OX42 and glial fibrillary acidic protein (GFAP) expression detection by Western blotting; the effect of microglia activation on astrocyte glutamate-glutamine cycle loop was studied through glutamine synthetase (GS) level detected by Western blotting.

In direct stimulation method, 10 μmol/L lead acetate and 100 μmol/L manganese chloride alone and co-exposure for 24 hours did not affect astrocyte activity and its GS expression. In conditioned medium method, 10 μmol/L lead acetate and 100 μmol/L manganese chloried alone and co-exposure for 24 hours significantly induced microglia activation. After C6 cells were cultured with astrocyte normal culture medium for another 24 hours, GFAP level was significantly higher in the exposure groups than in the control group. On the contrary, GS level was significantly lower in the exposure groups than in the control group. In co-culture methods, 10 μmol/L lead acetate and 100 μmol/L manganese chloride alone and co-exposure for 24 hours significantly induced microglia activation. Then the BV2 cells were rinsed gently and co-cultured with C6 cells for 24 hours, GFAP level were significantly higher in the exposure groups than in the control group. On the contrary, GS level was significantly lower in the exposure groups than in the control group.

Low-level lead or manganese alone or their co-exposure can cause microglia activation. The activated microglia can induce astrocyte activation and reduce its GS expression. Furthermore, compared with exposure alone, co-exposure had a synergistic effect.